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We have investigated mid-shaft stress fractures of the bowed femoral shaft (SBFs), well before the first report of an association between suppression of bone turnover and atypical femoral fractures (AFFs). Although all cases of SBF meet the criteria for AFF, SBFs can also occur in patients with no exposure to bone turnover suppression-related drugs (e.g., bisphosphonates). Using bone morphometry and biomechanical analyses, we devised a theory of AFF subtypes, dividing AFFs into fragility SBFs in the mid-shaft and "typical" subtrochanteric AFFs caused by suppressed bone turnover. The aim of this multicenter prospective study was to provide evidence for this novel concept in terms of biological activity.The study was conducted at 12 hospitals in Japan from 2015 through 2019. Thirty-seven elderly women with AFF were included and classified according to location of the fracture into a mid-shaft AFF group (n = 18) and a subtrochanteric AFF group (n = 19). Patient demographics and clinical characteristics were investigated to compare the two groups. The main focus was on histological analysis of the fracture site, and bone metabolism markers were evaluated to specifically estimate biological activity.All patients in the subtrochanteric AFF group had a history of long-term (3 years) exposure to specific drugs that have been reported to cause AFF, but 5 of the 18 patients in the mid-shaft AFF group had no history of exposure to such drugs. Femoral bowing was significantly greater in the mid-shaft AFF group (p 0.001). In the histological analysis, active bone remodeling or endochondral ossification was observed in the mid-shaft AFF group, whereas no fracture repair-related biological activity was observed in the majority of patients in the subtrochanteric AFF group. Levels of tartrate-resistant acid phosphatase-5b and undercaroxylated osteocalcin were significantly lower in the subtrochanteric AFF group (p 0.05).The possibility of our devised AFF subtype theory was demonstrated. Biological activity tends not to be suppressed in mid-shaft SBFs unlike in "typical" subtrochanteric AFFs involving bone turnover suppression. Although validation of the proposed theory in other populations is needed, we suggest that the pathology and treatment of AFFs be reconsidered based on its subtype.

Dentin matrix protein 1 (DMP1), a noncollagenous bone matrix protein produced by osteocytes, regulates matrix mineralization and phosphate homeostasis. The lack of a precise assay for circulating DMP1 levels impairs further investigation of the protein's biological significance. Because full-length precursor DMP1 is cleaved into NH2- and COOH-terminal fragments during the secretory process, we developed two new sandwich ELISAs for the NH2- and COOH-terminal fragments of rat DMP1. One of these ELISAs, ELISA 1-2, is based on two affinity-purified polyclonal antibodies against the DMP1-1 and DMP1-2 peptides of the NH2-terminal fragment, whereas the other, ELISA 4-3, is based on two affinity-purified polyclonal antibodies against the DMP1-3 and DMP1-4 peptides of the COOH-terminal fragment. The polyclonal antibodies were characterized in immunohistochemical and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) studies. Immunohistochemical analyses of rat bone using these polyclonal antibodies revealed DMP1 immunoreactivity in osteocytes and pericanalicular matrix, consistent with the previously reported osteocyte-specific expression of DMP1. LC-MS/MS analyses of rat plasma-derived immunoreactive products affinity-extracted with these antibodies revealed the presence of DMP1 in circulating blood. The ELISAs established with these antibodies met accepted standards for reproducibility, repeatability, precision, and accuracy. Circulating DMP1 and levels of other biochemical markers (osteocalcin, Trap5b, Dkk-1, and SOST) were measured in 2-, 4-, 8-, 12-, 18-, 24-, 72-, and 96-week-old Wistar male rats. Circulating DMP1 levels determined by ELISAs 1-2 and 4-3 significantly decreased with age. During rapid skeletal growth (2-12weeks), DMP1 levels measured by ELISA 4-3 were over three times higher than those measured by ELISA 1-2; however, DMP1 levels in old animals (72 and 96weeks) were almost the same when measured by either ELISA. DMP1 levels determined by both ELISAs were most highly positively correlated with the level of Dkk-1, second most highly correlated with the level of osteocalcin, and less highly correlated with the levels of Trap5b and SOST. These novel sandwich ELISAs for rat DMP1 are highly specific and allow precise measurements of circulating DMP1, which may be a new biochemical marker for osteocyte-mediated bone turnover.

Bone morphogenetic protein (BMP) is a potent inducer of ectopic bone formation, and TNP-470, a synthetic analog of fumagillin, is an antiangiogenic agent that strongly inhibits neovascular formation in vivo. We investigated the effects of TNP-470 on BMP-induced ectopic bone formation to clarify the role of angiogenesis in bone formation. Collagen pellets containing recombinant human BMP-2 (rhBMP-2) were implanted beneath the fasciae of dorsal muscles in mice. By daily subcutaneous administration of TNP-470, ectopic new bone formation was inhibited in a dose-dependent manner. Histological examination revealed that TNP-470 prevented proliferation of mesenchymal cells and chondrogenesis at the initial step of endochondral bone formation. Immunohistochemical staining with a specific antibody against bone morphogenetic protein type IA receptor showed that TNP-470 reduced the number of receptor-positive cells surrounding the BMP pellets. The inhibitory effect of TNP-470 on bone formation continued during the period of its administration, and discontinuation of treatment was followed by the resumption of the whole process of endochondral bone formation. This study showed that TNP-470 reversibly inhibits the biological activity of rhBMP-2 in the early stage of bone induction, suggesting that angiogenesis may play an essential role in the recruitment of BMP-receptor-positive cells that can respond to rhBMP-2 and differentiate into chondrocytes and/or osteoblasts.

The oxygen tension affects the function, differentiation, and transformation of various cells, including bone cells. In pathological conditions such as rheumatoid arthritis (RA), rapidly destructive arthropathy, and primary or metastatic tumors, severe bone destruction or osteolysis occurs. Abundant blood vessels are often observed around these destructive lesions. At such sites, we have confirmed the increased production of reactive oxygen species (ROS) induced by a high oxygen tension and/or oxidative stress, as well as numerous osteoclasts detectable by immunohistochemistry. These findings suggest that osteoclasts are influenced by the high oxygen tension in pathological bone lesions because the zone around blood vessels has a relatively high oxygen tension. In this study, we investigated the effects of oxygen tension on osteoclastogenesis by culturing human CD14-positive cells (osteoclast precursors) with or without osteoblast-like supporting cells (Saos-4/3 cells) under a normal oxygen tension (20% O(2)) or a high oxygen tension (40% O(2)). A high oxygen tension markedly prolonged the duration of osteoclast precursor formation in the presence of supporting cells, and also markedly and persistently increased the production of macrophage colony stimulating factor (M-CSF) by supporting cells. Furthermore, we found an increase of cells expressing M-CSF and cells positive for tartrate-resistant acid phosphatase (TRAP) in hypervascular destructive bone lesions of RA patients where ROS were also abundant.

The effects of estrogen on bone formation and bone resorption were examined in an experimental model of ectopic bone induction. In this experimental model, ectopic ossicles of uniform size were elicited reproducibly in three weeks by implanting pellets containing murine osteosarcoma derived bone morphogenetic protein (BMP), into the muscles of mice. Thereafter the induced ossicles showed a gradual reduction in bone mass due to negative bone balance. To estimate the effects of estrogen on bone formation and bone resorption, β-estradiol-3-benzoate was administered exogeneously to host mice from day 21 to day 41 (0.45 μg/g body weight, on alternate days, 10 treatments). Radiologic and histologic analysis confirmed the positive effect of estradiol on preserving bone mass over this period. Quantitative analysis showed an increase in the Ca content in ossicles after 21 days of estradiol treatment (139.1% of the baseline value on day 21). In the control group, the bone mass was reduced (47.4% of that on day 21). Bone resorption was determined by the reduction in 45 Ca radioactivity from ossicles prelabeled with this radioisotope before estradiol administration. The rate of loss of the prelabeled 45 Ca radioactivity was suppressed by estradiol (68.8% of prelabeled value in estradiol treated group and 87.7% in control group). The effect of exogeneous estrogen on the bone forming phase of bone turnover in the ossicles was quantified by incorporation of 45 Ca and 3 H-proline. These bone formation parameters were increased to 2.2 and 1.9 times higher than those of the control group. Based on these data, estradiol appears to preserve ectopically induced bone in mice, and this effect is derived mainly from enhanced bone formation and partly by suppression of bone resorption during bone turnover.

The effect of tumor necrosis factor alpha (TNF-alpha) on ectopic endochondral bone formation was studied in an experimental system for bone induction using murine osteosarcoma-derived bone-inducing substance. Ectopic new bone formation was inhibited by daily administration of recombinant human TNF-alpha (20-200 micrograms/kg body weight per day, intraperitoneally) after subcutaneous implantation of the bone-inducing substance into mice. Histological examination revealed that TNF-alpha prevented mesenchymal cells from differentiating into chondrocytes in the process of endochondral bone formation. The inhibitory effect of TNF-alpha continued during the period of its administration, but not after its administration was stopped. The bone induced in a three week period after discontinuation of TNF-alpha administration was histologically normal, but smaller than that induced in control animals. These findings suggested that TNF-alpha reversibly inhibits the biological activity of the bone-inducing substance or impairs the ability of cells to respond to the bone-inducing substance at an early stage of ectopic bone formation.

Fracture healing, which involves a cascade of biological tissue responses, may be affected by various biochemical substances. One of these substances is tumor necrosis factor alpha (TNF). Studies were made on the effects of TNF on healing of fractured ribs of rats. Fracture healing was inhibited by daily administration of recombinant human TNF (400 micrograms/kg body weight per day, intraperitoneally) after fracture. The rate of union on day 20 was significantly lower in the TNF-treated group (4/18, 22.2%) than in the control group (14/18, 77.8%) (p less than 0.001 by Chi-square test). Histological examination showed that TNF inhibited cartilagenous callus formation. On day 10, cartilage was seen in the gap zone and under the periosteum in the control group, but no cartilage formation was observed in the gap zone in 9 of 12 specimens from the TNF-treated group. On day 20, the fracture ends were united by newly formed bone in the control group, but mature fibrous tissue was seen in the gap zone, and bony or cartilagenous union was not achieved in the TNF-treated group. These results show that TNF inhibits cartilage formation in the early phase of bone induction in fracture healing and suggest that this effect of TNF is due to its inhibition of differentiation of mesenchymal cells into chondroblasts.